This invention relates to methods of testing human individuals for impaired immune function indicative of the presence of, or predisposition to, diseases associated with compromised immune competency. Such tests may be used, for example, diagnostically, prognostically, and as a guide in determining the need for preventive or therapeutic treatment for the disease or condition so indicated.
More particularly, the invention employs a surrogate measure of DNA repair activity based on serum/plasma thiol status as a biomarker of human health. Thus, the method of the invention involves the measurement of chemically reactive thiols present in naturally occurring amino acids, polypeptides and proteins found in human serum or plasma. The concentration of these thiols can predict DNA repair capacity and immune cell responsiveness, and they are therefore useful indicators of disease progression where impaired immune function is an essential component of the disease. HIV infection, AIDS, cancer and autoimmune disorders are examples of diseases that have immunological components.
European patent No. 0 229 674 as well as several recently published papers (Pero et al, Carcinogenesis 6:1055-58, 1985; Pero et al, Mutation Res. 142:69-73, 1985; Pero et al, Carcinogenesis 10:693-97, 1989; Pero et al, Carcinogenesis 10:1657-64, 1989) disclose that DNA repair in general, and specifically the quantitative estimation of adenosine diphosphate ribosyl transferase (ADPRT), is a useful endpoint to estimate health risks in the detection, prevention and treatment of human chronic age-associated diseases such as cancer, conditions that predispose to cancer, and autoimmune diseases. In another aspect, cellular ADPRT activity has been shown to relate to immune cell responsiveness (Scouvassi et al, Carcinogenesis 8:1295-1300, 1987; Pero et al, J. Neurosurg. 77:601-06, 1992; Johnstone and Williams, Nature 300:368-79, 1982; Johnson et al, Int. J. Biochem. 22:67-73, 1990), and both these parameters have been shown to be modulated by the cellular reduction/oxidation (redox) balance thought to be in turn mediated by the thiol containing peptide, glutathione (Pero et al, Cancer Det. Prevent. 14:555-61, 1990; Pero et al, Cancer Res. 50:4619-25, 1990; Fidelius et al, Exp. Cell Res. 170:269-75, 1987; Fidelius and Tsan, Immunology 61:503-08, 1987; Fischman et al, J. Immunol. 127:2257-62, 1981; Hamilos and Wedner, J. Immunol. 135:2740-47, 1985).
Glutathione exists in the millimolar range within cells (Kosower, Int. Rev. Cytol. 54:109-60, 1978; Meister, Science 220:472, 1983) and as such it is believed to be the primary cellular reductant protecting cells from oxidative cellular injury. However, glutathione levels in human serum/plasma (i.e. 2-27 .mu.moles/liter in Buhl et al, The Lancet, 1294-98, Dec. 2, 1989; Ayers et al, Anal. Biochem. 154:186-93, 1986) represent only a minor portion of the total reactive thiol groups present because the proteins in serum/plasma constitute the major source of reactive thiol groups (i.e. 113-133 .mu.moles/liter in Ellman, Arch. Biochem. Biophys. 82:70-77, 1959 and Ayers et al, Anal. Biochem. 154:186-93, 1986). Therefore, the art teaches that there are at least two distinct classes of thiols in serum/plasma and other biological tissues; namely protein thiols and nonprotein thiols. A review of the literature supports that conventional procedures for the analysis of serum/plasma thiols in relation to human health consequences are based on the analysis of nonprotein thiol sources such as glutathione or cysteine where protein thiols are excluded from the analysis by the assay procedure or removed by precipitation using agents such as trichloroacetic acid, metaphosphoric acid, sulfosalicylic acid or perchloric acid before any analysis of nonprotein thiols is undertaken (Beutler and Gelbert, J. Lab. Clin. Med. 105:581-04, 1985; Buhl et al, The Lancet, 1294-98, Dec. 2, 1989; Eck et al, Biol. Chem. Hoppe Seyler 370:101-81, 1989; Burgunder et al, Eur. J. Clin. Invest. 18:420-24, 1988; Burgunder and Lauterburg, Eur. J. Clin. Invest. 17:408-14, 1987; Mimic-Oka et al, Biochem. Med. Met. Biol. 39:48-54, 1988; Martensson, Metabolism 35:118-21, 1986; Vendemiale et al, J. Hepatology 9:359-65, 1989). Nonprotein thiol analysis of biological samples has evolved as the standard assay procedure principally because of the strong scientific belief that glutathione, a nonprotein thiol, is the primary cellular reductant protecting cells against the harmful health effects of oxidant injury (Meister, Science 220:472, 1983).
Oxidative cellular damage has been postulated to be an important factor in (i) ageing (Harmon, Age 7:111-31, 1984), (ii) diabetes (Wilson et al, Diabetologia 27:587-91, 1984), (iii) drug resistance (Spitz et al, J. Cell Physiol. 156:72-9, 1993), HIV+/AIDS (Baruchel and Wainberg, J. Leuk. Biol. 52:111-14, 1992), (iv) initiation and promotion of cancer (Marnett, Carcinogenesis 8:1345-73, 1987; Cerutti, Science 227:375-81, 1985), (v) etiology of cardiovascular and autoimmune diseases (Cross et al, Ann. Int. Med. 107:526-45, 1987) and (vi) modulation of immune function (Carson et al, J. Exp. Med. 163:746-51, 1986). Most of this evidence comes from evaluating oxidative stress by comparing glutathione deficient to glutathione proficient cells. For example, glutathione (or cysteine, its synthetic precursor) deficiency has been shown to (i) predispose cells to increased sensitivity to DNA damage (Edgren et al, Int. J. Rad. Biol. 40:355-63, 1985; Valis, The Lancet 337:918-19, 1991), (ii) inhibit DNA repair (Pero et al, Cancer Res. 50:4619-25, 1990; Edgren and Revesz, Int. J. Rad. Biol. 48:207-12, 1985), or (iii) induce immune cell response deficiency (Hamilos and Wedner, J. Immunol. 135:2740-47, 1985; Fischman et al, J. Immunol. 127:2257-62, 1981; MacDermott et al, Immunology 57:521-26, 1986; Droege et al, Immunobiology 172:151-56, 1986; Stacey and Craig, Experienta 45:180-81, 1989). In other words, the art teaches that the nonprotein thiol component is the important factor relating oxidative cellular damage to human disease development, and the protein thiol component, which quantitatively dominates in biological samples, has no direct or regulatory relevance to the health consequences of redox imbalance, and if it indicates anything at all, it is an indirect and nonspecific estimate compared to the major regulatory role of the nonprotein thiol component.
Additional evidence for this interpretation is taken from the medical literature where serum/plasma thiols have been employed to monitor health disorders. Malignant disease (Beutler and Gelbert, J. Lab. Clin. Med. 105:581-84, 1985), chronic renal insufficiency (Mimic-Oka et al, Biochem. Med. Met. Biol. 39:48-54, 1988), glucose mediated insulin secretion (Ammon et al, Diabetologia 32:797-800, 1989), ethanol ingestion (Burgunder et al, Eur. J. Clin. Invest. 18:420-24, 1988; Vendemiale et al, J. Hepatology 9:359-65, 1989), fasting (Martensson, Metabolism 35: 118-21, 1986), HIV infection (Buhl et al, The Lancet, 1294-98, Dec. 2, 1989), AIDS (Eck et al, Biol. Chem. Hoppe Seyler, 370:101-08, 1989), and cirrhosis (Burgunder and Lauterburg, Eur. J. Clin. Invest. 17:408-14, 1987) represent nearly all the medical conditions where serum/plasma thiols have been used successfully to monitor health disorders. In all cases, serum/plasma nonprotein thiols such as glutathione or cysteine were estimated, and great care was taken to eliminate protein thiols from the assay procedure. These data clearly indicate that it was not obvious to one skilled in the art to include serum/plasma protein thiols in the analyses, or that they might be indicators of the health consequences of oxidative stress, as good as, or even better than, the nonprotein thiols.
Congestive heart failure (Belch et al, Br. Heart J. 65:245-48, 1991) and rheumatoid arthritis (Pullar et al, Br. J. Rheumat. 26:202-06, 1987) are the only exceptions found in the scientific literature where both serum/plasma protein and nonprotein thiols were included in the final analyses. However, the logic behind these exceptions did not indicate that total serum/plasma protein and nonprotein thiols were a better indicator of the health consequences of oxidative cellular damage than were serum/plasma nonprotein thiols. Contrarily, it was postulated in these studies that because serum/plasma albumin was an important factor to these diseases, and because albumin is the major protein component of serum/plasma and contains numerous thiol functions, it followed that estimating total serum/plasma protein and nonprotein thiols was an effective surrogate measure of the oxidation state of albumin. Therefore, the inclusion in the serum/plasma thiol assay of nonproteins such as glutathione or cysteine and proteins other than albumin added no significant methodological advantage even though they were included in the final analyses and contaminated the estimation of albumin thiols.